In this project we determined major antigenic relationships of various rotavirus strains derived from humans and animals. The elucidation of the neutralization specificities of rotavirus is important in order to achieve a more comprehensive understanding of rotavirus epidemiology and for formulation of an effective strategy for vaccination. By using various single gene substitution human x human or human x bovine rotavirus reassortants and hyperimmune guinea pig antiserum raised against each reassortant, we successfully characterized the VP4 neutralization specificity of representative human rotavirus strains (Wa, DS-1, ST3, K8, and 69M) and established five distinct VP4(P) serotypes based on a >20-fold antibody difference criterion that is applied to classification of VP7 serotypes. Neutralization specificities on the VP4 capsid protein of two rabbit rotaviruses (C11 and Ala) originally isolated in the U.S. were found to be related antigenically in a two-way fashion to human rotavirus VP4 of VP7 serotype 6 PA169 strain (isolated in Italy) and of the VP7 serotype 8 HAL1160 strain (isolated in Finland).